1. Field of the Invention
The present invention relates to an analytical method, and more particularly to the analytical method of post-translational modifications in hemoglobin.
2. Description of the Related Art
Excess production of nitrogen monoxide (NO) is found in inflamed tissues activated by neutrophils and phagocytes. In endogenous oxidative metabolism, nitrogen monoxide is reacted with reactive oxygen species (ROS) to form reactive nitrogen oxide species (RNOx) such as peroxynitrite, which is a biological nitration agent, leading to the formation of 3-nitrotyrosine (3NT) on protein [1]. ROS and RNOx may also react with other biomolecules in cells to produce chemical modifications of different types, including deoxyribonucleic acid (DNA) and lipid [2-3]. Peroxynitrite is not the only source of 3NT in vivo, and peroxidase and certain metalloproteins [4] may also cause the formation of 3NT.
3-nitrotyrosine (3NT) in vivo has been detected under several pathological or physiological conditions. Inflammation and neurodegenerative diseases are correlated with nitration of tyrosine [5]. However, compared to protein phosphorylation, the nitration of tyrosine in cells may not be modified easily. In the normal content of 3-nitrotyrosine (3NT) in vivo, there is one 3NT in every approximately 106 tyrosine; and in an inflammation, there is one 3NT in every 104 tyrosine [5].
The nitration of tyrosine in post-translational protein directly leads to inhibition of protein functions or inhibition of tyrosine phosphorylation [6,7]. The nitration of tyrosine is not a permanent modification, and it can be reversed under redox control. Present literatures show that smokers and lung cancer patients have contents of protein nitration and oxidation in serum protein higher than those of normal persons [8,9], indicating that oxidation and nitration pressure play an important role in smoking and cancer development process [10]. Literatures [8,9] also point out that protein 3-NT can be quantified by western blotting, but this method does not provide high accuracy or specific measurement. The advancement of proteomics based on mass spectrometry can perform analyses by focusing on the identification of a specific nitrated protein and the position of a modified amino acid [11]. Since the nitrated tyrosine protein in vivo has a very low content, therefore the antibody of 3-NT is generally used to concentrate the content of protein including 3-NT before the western blotting or mass spectrometry takes place [12]. Other concentration methods including a series of reactions and derivatizations are developed extensively for the analysis of proteomics to overcome the problem of detecting a low content of nitrated protein [9, 13-15].